JPS6012975A - Microbiologically obtained d-2-hydroxy-4-methyl pentanoic acid-dhydrogenase and method for obtaining same - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The object of the present invention is to relate to a microbiologically obtained D-2-hydroxy-bovine-methylpentane e-dehydrogenase, which dehydrogenase contains a] coenzyme nicotinamide; -D associated with adenine dinucleotide (NAD”)
-2-Hydroxy-bovine-methylpentanoic acid dehydrogenation reaction, 2-keto bovine-methylpentanoic acid conversion, b
] In association with the coenzyme NAD+, it catalyzes the dehydrogenation reaction of other D-2-hydroxycardoic acids, converting them to the corresponding 2-ketocardioic acids, D-2-hydroxy-pentanoic acids, D-
Additive to 2-hydroxy-hexanoic acid, D-2-hydroxy-octanoic acid, D-2-hydroxy-(methylmercapto)-2-hydroxy-3-phenylzolopionic acid c) catalyzes the stereoselective reduction reaction of 2-keto bovine-methylpentanoic acid, together with NADH as a coenzyme, to D-2-hydroxy-bovine-methylpentanoic acid; d ) other 2-ketocardiacids, especially 2-ketobutyric acid, 2-
Ketopentanoic acid, 2-keto-3-methylbutyric acid, 2-ketohexanoic acid, 2-keto-3-methylpentanoic acid, 2-ketooctanoic acid, 2-keto-3-mercaptopropionic acid, 2-keto-(methylmercapto) ) catalyze the stereoselective reduction reaction of acetic acid and 2-keto-3-phenylzolopionic acid together with NADH as a coenzyme into the corresponding D-2-hydroxycardoic acid, and e) dehydrogenation reaction. Optimum pH range 7.5-9.0 for
f) having an optimum pH range of 5.59.0 for the reduction reaction; and g) having a pH-stable range of 3.5 to 10.

Another object of the present invention relates to a method for obtaining a new microbiologically obtained D-2-hydroxy-bovine-methylpentanoate-dehydrogenase, which method can be used to obtain a microbiologically obtained D-2-hydroxy-bovine-methylpentanoate dehydrogenase. Tal A
(I+actobacillus), 1uscasei ssp.
, pseudoplantarum ) (DSM 2
0008), Lactobacillus casei ss
p. alactosus ) (DSM 20020
), Lactonoζ Shiranu Kasei Nuzo Rhamnosnu (L
actobacillus casei ssp.

rhamnosus ) (DSM 2017 B)
, Leuconostoc oenos
oenos ) (DSM20252), l, Leuconostoc Lact.
is ) (DSM 20202) or Leuconostocmes
enteroides ) (DSM 20343)
containing carbon sources, nitrogen sources, vitamin sources, optionally growth factors, and inorganic salts and 6.5% at the beginning of breeding.
After breeding, the cells are obtained by centrifugation, and the cells are incubated at pH 71c.
It is characterized in that it is digested in a buffered suspension and the enzyme from the crude extract is further concentrated in a manner known per se.

Finally, the object of the present invention is to use novel enzymes to produce D-2-hydroxy-bovine-methylpentanoic acid, D-2-hydroxy-bentasiic acid, D-2-hydroxy- 2-ketocarginic acid corresponding to hexanoic acid, D-2-hydroxy-octanoic acid, D-2-hydroxy-(methylmercapto)-butyric acid or D-2-hyroxy-3-phenylsoropionic acid. Convert to 2
-Keto-methylpentanoic acid, 2-ketobutyric acid, 2-ketopentanoic acid, 2-kedo-3-methylbutyric acid, 2-ketohexanoic acid, 2-keto-3-methylpentanoic acid, 2-ketooctanoic acid, 2-keto- 3-mercaptopropionic acid,
It is also relevant to convert 2-keto-(methylmercazoto)-butyric acid and 2-keto-3-phenylzorobio>et into the corresponding D-2-hydroxycarzenic acids.

PRIOR ART For the stereospecific reduction of 2-ketocardoic acid to D-2-hydroxycardoic acid, the following enzymes related to NADH are known from the literature: D-D-lactate telodrogenase E, 0. 1.1.1.28). The new enzyme is
Since D-2-hydroxy-bovine-methylpentanoate-dehydrogenase does not accept pyruvate as a substrate, D
-Distinguished from lactate dehydrogenase. Furthermore, D-2-
Foxy fatty acid dehydrogenase (E, 0°1.
1.1. ! ; l 8 ) was isolated from rat kidney, in which it was found that long-chain fatty acids β-
Involved in oxidation. However, for this enzyme, 2
- Only hydroxystearates are described as substrates.

Function: The enzyme according to the invention can be used as described in the Deutschen Sammlung Huon Microorganism in Göttingen.
Sammlung von Mikro
organizations in G.
Various microorganisms, namely Lactopacillus casei Suji pseudoplantarum (L')
obacillus casei ssp, pseudo
oplantarum ) (DSM 20008),
Lactopasillanu casei Suji Rhamnosnu (Lact
obacillus casei ssp, rhamn
osus) (DSM 20178), Lactobacillus casei
lus caseissp, alactosus)
(DSM 20020), Leuconostoc oenos (DSM
2025 '2), or Leuconostoc mesentero
ides) (DSM 20343) and Leuconostoc Lacti
s ) (DSM 20202). These strains are Lactopacillus (Lactob
acillus) and Leuco
D-2-hydroxy-4-methylpentanoic acid-
Identified as a dehydrogenase-producer.

The new enzyme is measured by a photometric test. The test formulation (3.0 OrnlV) contains 0.1 molar phosphate buffer pH 7.0, 0.235 mmol NADH, 0.79 mmol 2-keto-bovine-methylpentanoic acid and limited amounts of enzyme. NADH - decrease in absorbance at 340 nm
When measuring at 30 DEG C., the zero value obtained when the test formulation is incubated with 2-ketocarzenic acid;'zL is taken. Enzyme activity is stated in international units (U), where IU is NADHIQ-a/mm, d
catalyze the amount used. For calculations, NADH
The extinction coefficient of 1 mole of is 6.22 at 340 nm. lO
is based on. For the test formulations listed,
Both D-2-hyroxybovine-methylpentanoate-dehydrogenase and L-2-hydroxy-4-methylpentanoate-dehydrogenase are captured. The Zoranu strain is
It is tested again with chiral D-2-hydroxy-bovine-methylpentanoic acid as a substrate not available on the market. D
D- according to the invention compared to -2-hydroxycarinic acid
Only 2-hydroxy-bovine-methylpentanoate-dehydrogenase shows activity, L-2-hydroxy-bovine-methylpentanoate-dehydrogenase is inactive. Another proof for the stereospecificity of the catalytic transformation comes from tests in membrane reactors, which
Further details are provided below.

The specific and volumetric activities determined in the crude extracts of the microorganisms producing the individual D-2-hydroxy-bovine-methylpentanoate-dehydrogenases are listed in Table 1. Breeding of microorganisms is carried out by Deutsche, Sammlung and Microorganisms.
n Sarnmlungvan Mikroorgan
The experiments were carried out in the medium described in the catalog of Ismen). Activity measurements using two other 2-ketocardanoic acids as substrates revealed that Lactobacillus casei nup pseudoplantarum (Ll.
us casei ssp.

pseudoplantarum ) (DSM 20
showed that the enzyme from 008) has the broadest substrate spectrum and is therefore particularly suitable for converting a series of D-2-hydroxycarzenoic acids with good activity (see Table 1). ).

Next, the new enzyme was isolated from Lactopanlas casei nup pseudozolantarum (Lactobacj-11usc).
asei ssp, pseudoplantarum
) (DSM 20008) are exemplarily described: 1. Breeding of microorganisms For breeding, Lactobacillus casei pseudoplantarum (Lactobacillus casei
i ssp, pseudoplantarum)
(DSM 20008) is cultured in the following media: Glucose 2 20g Yeast extract 10g Meat juice 0.5g Sodium acetate 5g K2HP042g MgSO40,2g MnSO40-05'' deionized water until 11.

The pH value of this solution is adjusted to 6.5 and the solution is further sterilized at 121° C. (2 Parr) for 15 minutes. For breeding, Lactopanrus casei, xzo pseudozolantarum (
Lactobacillus casei ssp, p
seuaoplantarum) by puncture agar-tubules and incubated at 30° C. for 16-20 hours.

Next, the grown culture 4- was used as the inoculated culture and culture medium 20
For 0rd (500-Erlenmayer (Erl
enmeyer)-in a flask). 20~
After 24 hours, this 200- can be used as a preculture for the 101 fermenter and can then optionally be inoculated with 2001 and 500 (M). The pH value is maintained in the fermenter with concentrated ammonia at 5.0 O. Cultivation in the fermenter is carried out under gentle stirring and with nitrogen gas blowing.

At the end of the logarithmic growth phase, the culture is cooled and the cells are harvested by centrifugation. In this case, a centrifuged cell mass of 45 kg is obtained from culture 4-500A. This biomass can be intermediately stored for 2.3 months at -20°C without significant loss of activity.

2. Isolation and purification of enzyme a] Crude extract Lactobacillus casei ssp.

pseudoplantarum ) (DSM 20
008) - 500 g of wet mass are suspended in 50 mlj mol of phosphate buffer pH 7.0 containing 0.1 (v/v)% 2-mercazotoethanol. 125o
- final volume corresponds to 40% cell suspension.

Cells are disintegrated in a continuously working glass bead-ball mill or in a high-pressure homogenizer.

In this case, Dynomuhle type KDL manufactured by Bachofen is used as the galanutama-seelmilt.

A grinding container with a content of 600 m1 requires 0.25 to 0.5 mm.
filled with glass beads of size, therefore 510 m
A shaking capacity of 1 is obtained (85%).
It is carried out at a stirring shaft rotation speed of rpm and a reflux rate of 51/h. The cooling jacket and agitation bearing of the grinding vessel should be at least 120 degrees centigrade to sufficiently prevent heating of the product during the process.
Cooled in ethylene glycol solution at °C. After three passes, a degradation rate of >90% has been achieved. Suspension pH
The value is reduced to 6.1 during homogenization and adjusted to pH 7.0 with caustic potash.

As a high-pressure homogenizer, for example, Manton Gaulin type 15M-8T
Equipment of A (A, P, V, 5chr; der GmbH,
Lubeck) can be used. In this case, the cell suspension is degraded specifically for microbial degradation and a reflux rate of 54 A/h using a homogenization valve at 550 Parr. After two passes, a decomposition rate of >90% is achieved. After all passes, the homogenate has 2
To prevent heating of the product above 0°C, it is cooled to a temperature below +10°C by means of a reflux condenser. Suspension pH
The value is pH 7, O 1l after decomposition? l: Adjusted.

b) Liquid-liquid distribution According to the first distribution process, the cell debris from the crude extract is
It should be separated by the description in West German Patent Specification No. 2,639,129. For this, an aqueous two-phase system is obtained, this two-phase yarn is composed of polyethylene glycol 1500
20 (w/w) %, pH 7.0 phosphate buffer 5
(w/w)% and 1250 rnl of crude extract 2.
Contained in 5 kg of system. To achieve distribution equilibrium, the two-phase system is stirred for 1 hour and subsequently separated by centrifugation. Large volumes can be continuously produced, e.g. by α-Laval's Gyro
rotester) type or Huenuto 77 rear (Wes
tfalia), preferably in a dish separator of type 5AOH-205. The upper phase (14-40a) actually has the total activity of D-2-hydroxy-bovine-methylpentanoate-dehydrogenase (yield 99 cg). The lower phase contains cell debris and is discarded. The enzyme-containing upper phase is 2880 m7! Polyethylene glycol 10000 2 (v/v)%, pH 5.0
of phosphate buffer and 0.4-mole of sodium chloride are added and stirred for 1 hour. The polyethylene glycol/salt system that forms is deposited in a vertical cylinder and the separation is complete after about 1 hour. For this distribution process,
D-2-Hi rI Coxy-Bovine-Methylpencunate-dehydrogenase is extracted into the salt-containing lower phase. Separation of the phases takes place by discharging the lower phase.

The diafiltration lower phase was concentrated in an Am1con hollow fiber cartridge CHIP IO type) and I]H7°0
Diafiltrate to 50 mmol concentrated salt by adding 7JO of phosphate buffer.

d) DEAF-cellulose-chromatography The concentrated and diafiltered enzyme was
tman) - delivered on a 5x30 cm column packed with cellulose DE 52 in a K drenzo. D.E.
AE-cellulose was prepared in 50 mmol of phosphate buffer at pH 7.0 and 0.1 (v/v) of 2-mercazotoethanol.
v) is equilibrated against a buffer containing %. The column is first post-washed with starting buffer approx.
The enzyme is then added to the starting buffer with 0 to 0.2 sodium chloride.
Elute with a 5 molar linear gradient (2×27). D-2-Hydroxy-bovine-methylpentanoate-dehydrogenase is eluted with approximately 0.1 mole of sodium chloride. Active distillation, concentrated by ultrafiltration and stored at Bovine °C. The purification process is summarized in Table 2.

Table 2: Purified crude extract of D-2-hydroxy-bovine-methylpentanoate-dehydrogenase 1250 50500 18625 0°6
1 100 + upper phase 1 1440 6420 184
38 3.01 99 4.9 Department of Engineering fl 106 [1
3795158574,18856,9 Diafiltration 18
7 3150 15064 4.78 817.8DE
AE-4934244054524,875740,8
The enzyme preparation obtained by the cellulone last step can be used for industrial purposes. This enzyme can be used with other chromatographic methods, e.g.
ite) OG 50 ion exchange chromatography, Sephacryl S 2
00 gel filtration or Phenylcephalone (5eph
arose ) can be further purified by hydrophobic chromatography on OL-4B to a specific activity of 110 U/+++9.

The substrate spectrum is not changed thereby.

The molecular weight of D-2-hydroxy-bovine-methylpentanoate-dehydrogenase was determined by the method of Antriuse ("Me
th, Biochemical Analysis”,
73000±1000 by ultrafine gel filtration with 5ephacryl S 200 according to Vol. 18, pages 1 to 53 (1970).
0 Daltons are measured. As calibration substances, dequinutraso blue, ferritin, aldolase, bovine serum albumin, chymotrypsinogen A and cytochrome C are used.

D...2-Hydroxy-bovine-methylpentanoate-dehydrogenase according to the invention (can be used to obtain D-2-hydroxycarmenic acid on the d,,- side and L-amino acid-dehydrogenase on the other side) Together D-
It can be used to convert 2-hydroxycarboxylic acids to the corresponding optically active L-amino acids. Furthermore, the new enzyme can also be used to measure the concentration of 2-ketocarzenic acid or D-2-hydroxycarzenic acid converted as a substrate in an enzymatic process in aqueous solution. If the substrate concentration is small compared to KM, the reaction rate is -
depends on the substrate concentration in an order of magnitude. The measurement is extremely simple to carry out, and the change in absorbance of NADH is determined by 34 Q
The concentrations tracked in nm and further tested are read from the corresponding calibration curve.

Stereoselectivity of enzymatic conversion was examined in an enzyme membrane reactor. In this case, for the regeneration of NADH used in reducing 2-ketocalic acid, formate dehydrogenase is used in a sodium formate buffer and the natural NADH
NADH covalently bonded to ethylene glycol (average molecular weight 20,000) is used (prepared as described in West German Patent Specification No. 28 41 414). Individually converted: 2-ketoprotein-methylpentanoic acid (nato-1 salt),
2-Keto-(methyl mercapto-butyric acid (sodium salt) and phenyl hyaluric acid (sodium salt)). The product stream leaving the membrane reactor is continuously analyzed in a polarimeter. The resulting signal was evaluated on a calibration curve absorbed with an optically active 2-hydroxy acid.

Table 3 shows that under the selected conditions significantly high conversion rates are achieved; only the D-isomer of the 2-hydroxycarboxylic acid is observed. Furthermore, the results show that the enzyme according to the invention accepts an extended molecular weight NADH than the substrate.

Table 3; Reduction of 2-ketocarinic acid 2-keto-methylpentanoic acid -0.0478 D
7.0 mmol (100%) Example The invention will now be explained in more detail with examples; Example 1: D-2-Hydroxy-cow-methylpentanoic acid-
Dehydrogenase - Cleaning screening for producers
Microorganinum (DeutschenSamm)
(Lung von Mikrooganismen)
Lactopacillus (Lactobacillus) from the stockpile of
llus ) and Leuconostoc ( Leuconostoc )
The experiment was carried out using 45 types of bacterial strains of the stoc class. These strains were bred in a shaking flanco in the medium described in the DSM catalogue, and the cells were removed after 20-25 hours and subsequently disintegrated by treatment with ultrasound. Insoluble cell fragments were centrifuged and the clear top was used as the crude extract. Enzyme activity was determined in a photometric test, where the test formulation (3.OOmJ) had the following composition:
NADH 0. 2 3 5 mmol, 2-keto bovine-methylpentanoic acid 0.79 mmol and pH 7.0
of phosphate buffer 0. 1 mole. This test formulation was heated at 30°C.
After incubation for 10 minutes, the reaction was started by adding the limiting amount of enzyme.

Enzyme activity was measured at 340 nm by decreasing the absorbance of NADIH. If a zero value is taken, it was obtained when starting the test without 2-keto-bovine-methylpentanoic acid. Enzyme activity is stated in international units, where lU is NADH per minute and l-
Means the use of 1 micromolar.

The described tests do not allow us to report at all what stereospecificity the NADH-dependent 2-hydroxy-bovine-methylpentanoate dehydrogenase has. Such a program was implemented in the manner described above.

This is because both D-specific dehydrogenases and L-specific dehydrogenases were important.

The difference between the activities of both these is possible by following the conversion of D-2-hydroxy-10-methylpentanoic acid to NAD+, which NAD+U, D-2-hydroxy-cow-methylpentanoic acid- Catalyzed only by dehydrogenase, L-2-hydroxy-bovine-methylpentanoate-dehydrogenase is inactive with this substrate.

For that purpose, the following test formulation was used: NAD 3
．． 0 mmol, D-2-hydroxy-bovine-methylpentanoic acid 6.3 mmol and pHa.

of phosphate buffer 0.1M. This test formulation (-3°0
07] was incubated at 30° C. for 10 minutes, after which the reaction was started by adding the limiting amount of enzyme. If we take the zero value, it converts the K experiment to D-2-hydroxy-
Obtained when starting without bovine-methylpentanoic acid. Table 1 above lists six strains containing the enzyme D-2-hyroxybovine-methylpentanoate-dehydrogenase according to the present invention. , the kinetics of enzyme-catalyzed transformations with other 2-ketocarboxylic acids have been described. Enzyme activity is 2
- Keto bovine - Determined in the above test formulations by replacing in each case methylpentanoic acid by another keto acid as listed in Table 1. In this case, Lactopasillus casei streak pseudoplantarum (L.
actobacillus casei ssp.

pseudoplantarum ) (DSM 20
008) showed the broadest substrate spectrum.

Example 2: Relationship between reaction rate and pH value of enzyme-catalyzed conversion of 2 to D-2-hydroxy-4-methylpentanoic acid in the presence of D-2-hydroxy-bovine-methylpentanoate-dehydrogenase. The kinetics of the reduction of -keto-methylpentanoic acid was investigated in relation to the pH value of the reaction solution. The test formulation (3,00m) had the following composition:
NADHo, 1 mmol, 2-keto bovine-methylpentanoic acid 07 mmol, limiting amounts of enzyme, 0.1 molar buffers of various pH values as described in FIG.

In FIG. 1, the reaction rate (expressed as enzyme activity (U/mJ)) is described as a function of the pH value.

The enzyme exhibits a relatively wide pH-optimum between pH 5.5 and pH 7 for the reduction reaction.

The reaction rate of dehydrogenation of 2-hydroxy-bovine-methylpentanoic acid to 2-keto bovine-methylpentanoic acid catalyzed by D-2-hydroxy-bovine-methylpentanoate-dehydrogenase is similarly dependent on the pH value. was examined in relation to

The test formulation (3,OOmJ) had the following composition: 4.5 mmol NAD, 12 mmol D,L-2-hydroxy-bovine-methylpentanoic acid, limiting amount of enzyme, 2.
0.1 molar buffers of various compositions as described in the figure.

The kinetics of the dehydrogenation reaction (expressed as enzyme activity (u/mJ)) shows an optimum value within the range of pH 8 to IH9. Example 3: D-2-Hydroxy-bovine in relation to pH number - Storage stability of methylpentanoate dehydrogenase D-2-Hydroxy-bovine-methylpentanoate dehydrogenase at 0.03 molar in 0.1 molar buffers of various compositions
The cow was kept at constant temperature for 7 days at a protein concentration of m9/-. Thereafter, the activity of more residues as described in the example of cattle was measured. In this case, a particular pH stability was exhibited in the range from pH 3.5 to pH 1 o.o. After 24 days, still active >95
% could be detected.

Example 4: Relationship between reaction rate and substrate concentration The reaction rate when reducing 2-keto-cow-methylpentane 1tfD-2-hydroxy-cow-methylpentanoic acid,
The relationship with the concentration of the coenzyme NADH was examined in the following test formulations: phosphate buffer pH 70 01 molar, 2-
Keto bovine - 0.7 mmol of methylpentanoic acid, limiting amount of enzyme [DIAI - cellulose - chromatographically concentrated preparation, see Table 2]; in the test formulation (
7) NADH-Concentration Ha, 0.003~0.33 Mi!
It varied within a range of J%. The optimal reaction rate is 010
It has been shown that millimoles can be achieved. NADH no KM
- value is 0.0010 mmol.

NADPH cannot compensate for the coenzyme, NADH, which is 0.2 mi! Test concentrations up to JMole were tested.

The reaction rate when reducing various 2-ketocarboxylic acids is
It was also examined in relation to the concentration of keto acids. To that end, the following test formulations were used: 1) 0.1 mol of H7,O phosphate buffer, 0.1 mmol of NADH and a limited amount of enzyme (DEAE-cellulose-chromatographically concentrated preparation) , see Table 2). The concentration of the ketocarboxylic acid was varied in each case within the range from 0.01 mmol up to 60 mmol. Initial reaction rate, 340 nm/mi
The change in absorbance with n was estimated by nonlinear regression of the Mivaelis-Menten equation.

The measured kinetic constants KM and ■rnaX are summarized in Table 4.

Cow Table: Lactono Shiranu Kasei Nupu Shu 1
Plantarum (Lactobacillus case)
i ssp.

Substrate specificity of D-2-hydroxy-bovine-methylpentanoate-dehydrogenase from Pseudoplantarum ) 2-ketobutyric acid bovine 5.9 1.7XIO-'2-ketopentanoic acid 59.1 1.1X10-'2-'rl Hext>acid 58.4 1.1xlO-'2-ketooctanoic acid 54.0 3. lX10-'2-keto-3-methyl-butyric acid 16.9 4.8XIO2-keto-3-1
fru d > l > acid 26.2 2.2 X 10
- '2-Keto-methyl-pentanoic acid 27.4
6.0X10-52-keto-(methylmercaptoco-butyric acid 82.1 6.2XIO-'2-keto 3-mercaptopropionic acid 104.5 4.0X10-'
2-keto 3-phenylzolopionic acid 304.4 1.
The relationship between reaction rate and NAD'' concentration in dehydrogenating 5XlO''D-2-hydroxy-bovine-methylpentanoic acid was examined in the following test formulations: phosphate at pH 8.0. 0.1 molar buffer, 12 mmol D,L-2-hydroxy-bovine-methylpentanoic acid, limiting amount of enzyme. NA
The D-concentration was varied in the range from 0.10 to 10 mmol and the increase in absorbance due to the NADH produced during the reaction was measured at 340 nm. The optimal conversion is 4.5 mi!
It has been shown that this is achieved at a concentration of J molar. N.A.D.
The KM value of + is 0.45 mmol. NADP
cannot compensate for the coenzyme NAD+, which is
Test concentrations up to 10 mmol were tested.

The relationship between the reaction rate in dehydrogenating D-2-hydroxycarboxylic acid and the concentration of various 2-hydroxycarboxylic acids was examined in the following test formulations: phosphate at pH 8.0. Buffer 0.1M, NAD+4.5 Mi! J mole and limiting amount of enzyme. The concentration u of D-2-hydroxycarboxylic acid varied within the range from 0.1 mmol up to 30 mmol.

When no chiral D-2-hydroxycarzenic acid was used, the racemic compound was used. NADH produced during the reaction was measured at 340 nm. The initial reaction rate was estimated by the Miberellis-Menten equation, and the kinetic constants VmaX and KM were determined by nonlinear regression. The measured kinetic constants are summarized in Table 5.

Table 5: Lactobacillus casei
ssp.

D-2-hi)-1 from Pseudoplantarum).

Substrate specificity of methylpentanoic acid dehydrogenase, L-2-hydroxypentanoic acid 10.8 1.
4-XIO-'D, I, -2-hydroxy-hexanoic acid 11.3 9.3X10-'D, L-2-hydroxy-octanoic acid 10.0 6.2X10-'Example 5: Lactopacillus casei Nu. Zo Pseudobranchrum (L
actobacillus caseissp, pse
udoplantarum ) (DSM 20008
Stereospecificity of the reduction of 2-keto-methylpentanoic acid and other 2-ketocartaic acids using dehydrogenases according to the invention from N bound to &IJ ethylene glycol is expanded in molecular weight in an enzyme membrane reactor.
This is possible while using ADH. pH: G, NADH
is obtained as described in West German Patent Specification No. 2,841,414.

The modified coenzyme and the enzyme used, formate dehydrogenase (Kroner et al., T. Ohem, Tec.
h.

Biotechnol,” Volume 32, Page 130~
137 (1982)] and D-2-hydroxy-bovine-methylpecunate-dehydrogenase, using an ultrafiltration membrane YM5 (Amicon (Wit)).
(product of Ten) Co., Ltd.) K is retained in the reactor and contains the low molecular weight components of the reaction solution, the optically active 2-hydroxycarboxylic acid formed, the substrate (optically inactive ketoacid) that may not be converted, and the buffer used. Liquid is continuously removed from the reactor. The reactor volume is kept constant by dosing the keto acid and sodium formate buffer from the reservoir in equal proportions, in which case the rhizoid filtration leaves the reactor. The reactor volume is 10- and the concentration of PEG-NADH is 0.2
0 mmol, D-2-hyProxy-bovine-methylpentanoate-dehydrogenase GTR (27 U/mg) and 30 U of formate dehydrogenase were fed into the reactor. The substrate solution was sodium formate buffer 3 with a pH of 7.0.
00 mmol and corresponding 2-ketocalcimenic acid 7.0
Contained mmol. The reaction solution was continuously pumped through the membrane at 30 td/hour using a hose pump. Approximately 3 liters of ultrafiltrate were obtained every hour.

Continuously rotate the ultrafiltrate (perkin)
241]. Measure 463n
The experiments were carried out at 25°C. The concentration of the product was determined from the measured optical rotation using a calibration curve. This calibration curve is p)(
Detection was performed with an optically active 2-hydroxycarboxylic acid in 300 mmol of 7,0 formic acid buffer. For that purpose, the following preparation was used: L-phenyllactic acid CS
igma, Munchen), L-2-hydroxy-bovine-methylpentaic acid CSigna, MunCh
and D-2-hydroxy-bovine-(methylmercapto)-W acid CDegussa, Hanau). The reaction in the membrane reactor was followed for 15 hours. The steady-state values obtained are summarized in Table 3 above.

The substrate 2-keto Φ-methylpentanoate was used to test the stereoselectivity of 2-hydroxy-Φ-methylpentanoate-dehydrogenases produced by other microorganisms also listed in Table 1 above. In this case, the crude extract according to Example 1 was used as the enzyme source. Only the formation of D-2-hydroxy-bovine-methylpentanoic acid is 96-100
% conversion was observed.

[Brief explanation of the drawing]

FIG. 1 is a graph showing buffers of various compositions and various pH values, and FIG. 2 is a graph showing buffers of various compositions. Continued from page 1 0 Inventor Mariaregina Kula, Federal Republic of Germany Wolfenbütel Värstve Eng.・Biotechnologische Forschung Mituto Beschlenkter Haflang (G.B.F.) Braunschweig Steckheim, Federal Republic of Germany, Massierroeder V-Technology 1

Claims (1)

[Scope of Claims] l In microbiologically obtained D-2-hydroxy-bovine-methylpentanoate dehydrogenase, a) D-2 related to the coenzyme nicotinamide-adenine dinucleotide (NAD''); -catalyze the dehydrogenation of hydroxy-bovine-methylbetanoic acid to 2-keto-4-methylpentanoic acid, and b) dehydrogenation of other D-2-hydroxycarboxylic acids in conjunction with the coenzyme NAD+. was catalyzed to give the corresponding 2-ketocarboxylic acid, D-2-hydroxycarboxylic acid, D-
2-Hydroxy-hexa: exhibits additive activity towards [,D-2-hydroxy-ocmercabuto)-butyric acid and D-2-hydroxy-3-phenylpropionic acid, Q) 2-keto-4-methyl d) Stereoselective reduction of pentanoic acid to D-2-hydroxy-4-methylpentanoic acid by catalyzing the stereoselective reduction reaction of pentanoic acid together with NADH as a coenzyme; d) Stereoselective reduction of other 2-ketocarboxylic acids. catalyze the reaction together with NADH as a coenzyme to the corresponding D-2-hydroxycarboxylic acid; e) an optimal pH range of 7.5-9.0 for the dehydrogenation reaction;
and f] the optimum pH range for the reduction reaction from 5.5 to 7.0.
and g) a pH-stability range of 3.5 to 10.
Hydroxy-bovine-mef) Lrdntanoate-dehydrogenase. In the method for obtaining -4-methylpentanoate dehydrogenase, Lactobacillus casi
ei ssp, pseudoplantarLL, i)
(DSM 20 '008), Lactobacillus casei
caseissp, alactosus) (
DSM 20020), Lactobacillus casei nupu rhamnonunu
i ssp, rhamnosus) (DSM201
78), Leucononotoku Oeno, 2 (Leucon
ostoc oenos ) (DSM 20252
), Lθucon08tO
QLactis) (DSM 20202) or Leuconostoc mesenteroides (Leuconost.
ocmesenteroides ) (DSM 20
343 cells were bred in a liquid medium containing carbon sources, nitrogen sources, vitamin sources, optionally growth factors, and inorganic salts and having a pH value of 6.5 at the beginning of breeding, and after the end of breeding, the cells were are obtained by centrifugation, and the cells are p
Microbiologically obtained D-2-hydroxy-cow, characterized in that it is digested in a suspension buffered with H,-7 and the enzyme from the crude extract is further concentrated in a manner known per se. −
Method for obtaining methylpentanoate-dehydrogenase.